A facile fabrication and conjugation approach for biopolymeric-zwitterionic hydrogel microparticles toward accurate protein quantification using replica molding and peptide ligand conjugation

초록

Accurate and selective protein quantification is critical for early disease diagnosis. However, existing technologies and assay platforms often face limitations such as low probe titers, non-specific adsorption, and complex multistep manufacturing processes. In this paper, we introduce a straightforward and reliable replica molding technique to fabricate shape‒encoded hydrogel microparticles. These microparticles consist of potent biopolymeric conjugation handles and zwitterionic building blocks, which address key challenges in protein quantification. More specifically, photoinduced crosslinking of aqueous mixtures containing the polymerizable zwitterionic monomer 2‒methacryloyloxyethyl phosphorylcholine (MPC) and the amino polysaccharide chitosan (CS) is performed. The mixtures are confined within microwells and covered with hydrophobic wetting fluid that includes a photoinitiator and produces biopolymeric-zwitterionic hydrogel microparticles. This process, based on interfacially initiated free radical polymerization (IIFRP), ensures reliable shape replication, chemical reactivity, and super‒hydrophilic properties. Studies on crucial fabrication parameters by fluorescent labeling show the method's simple, tunable and robust nature. The conjugation of peptide ligands using carbodiimide chemistry and the binding assays for target proteins (interleukin 1β) demonstrate the efficient and highly selective nature of our fabrication-conjugation approach. This approach, combined with the biopolymeric-zwitterionic hybrid hydrogel microparticles, should enable the ready expansion to a wide range of promising zwitterionic building blocks and peptide ligand probes. Together, these advancements pave the way for tunable and accurate protein quantification. STATEMENT OF SIGNIFICANCE: Selective and reliable protein quantification assay is crucial in early disease diagnostics yet faces critical challenges. In this report, we present facile routes to generate highly selective microparticle suspension array platforms consisting of superhydrophilic zwitterionic building blocks and a potent aminopolysaccharide chitosan as an efficient conjugation handle. Simple carbodiimide chemistry-based conjugation of peptide ligands with specific affinity for a clinically important IL-1β target protein shows highly selective nature, while the protein binding assay further confirms the ideal binding characteristics illustrating several desirable performance features. We believe that the work described in this report is not only at the core of the biomaterial science field but also an important topic applicable to a wide range of areas including biosensing, biomedical engineering and disease diagnostics. Copyright © 2025. Published by Elsevier Inc.

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